The present proposal examines the hypothesis that orotic acid induces liver tumor promotion by creating differential mito-inhibitory environment in the surrounding liver while permitting the initiated hepatocytes to respond to growth stimuli to form hepatic foci and nodules. During the past grant period, it was observed that orotic acid is mito-inhibitory to hepatocytes both in vitro and in vivo. Further, hepatic nodules appear to be relatively resistant to the mito-inhibitory effect of orotic acid. Based on these recent preliminary observations the following three specific aims are proposed. (1) To determine the site at which orotic acid exerts its mito-inhibitory effect; (2) to determine the mechanism by which hepatic nodules become resistant to the mito-inhibitory effect of orotic acid; and (3) to determine whether orotic acid induced mito-inhibitory environment when coupled with a liver cell proliferative stimulus will exert a rapid promoting effect. In the first specific aim we will determine whether ribonucleosidedliphosphate reductase (RNR) is one target site of the mito- inhibitory effect of orotic acid. The effect of orotic acid on the regulation of the activity and expression of RNR will be examined both in vitro in isolated primary hepatocytes exposed to EGF and in vivo in the liver following partial hepatectomy (PH). The inhibitory effect of orotic acid will be correlated with the imbalances in nucleotide and deoxynucleotide pools induced in hepatocytes by orotic acid. The second specific aim examines the question whether the resistance of hepatic nodules to the mito-inhibitory effects of orotic acid is due to a defect in the (i) uptake and/or metabolism of orotic acid to uridylic acid and/or (ii) due to an altered RNR i.e. less sensitive to the effects of imbalances in nucleotide/deoxynucleotide pools. Both in vitro experiments using hepatocytes from nodules and in vivo experiments using rats bearing nodules will be employed. In the third specific aim, rats will be initiated with diethylnitrosamine and subjected to orotic acid induced mito-inhibitory environment coupled with PH to determine whether such a protocol selectively and rapidly promote the growth of hepatocytes to form foci and hepatic nodules. Since nucleotide pools and RNR are normal cellular constituents, it was tempting to speculate that orotic acid model of liver tumor promotion has the potential to be extended to other organs as well.